The present invention generally relates to methods for in vitro synthesis of ribosomes. More specifically, the present invention relates to methods of synthesizing, evolving, and screening ribosomes for variants.
Escherichia coli ribosomes are capable of polymerizing amino acids into complex polypeptides with diverse functions. To engineer or modify ribosomes, we have previously reported on the integrated synthesis, assembly, and translation (iSAT) system, in which ribosomal RNA (rRNA) can be in vitro transcribed and assembled into functional ribosomes. Here we report the coupling of the iSAT system with ribosome display, a method for stalling ribosomes, to create the ribosome synthesis and evolution (RISE) method. RISE uses mutated DNA to build a library of ribosomes that can then be screened for functionality under different conditions. With our optimized protocol, we observe >1,000-fold specificity for functional ribosomes, which allows for rapid screening of large libraries of rRNA mutations. As a demonstration, we used RISE to explore mutations of the ribosomal peptidyl transferase center, and found RISE rapidly converged libraries of 4,096 and 1.7×107 sequences back to the wild type sequence. Additionally, we evolved resistance to the antibiotic clindamycin and uncovered novel resistant combinations of base mutations. Moving forward, RISE will serve as a powerful new approach for exploring the effects of rRNA mutations on ribosome function and to ultimately isolate ribosomal variants with altered functionalities.
The applications of the disclosed methods include in vitro study of ribosome biogenesis; ribosome evolution for ribosome engineering or modification; encapsulation within emulsions for compartmentalized ribosome evolution; rapid, high-throughput testing of new antibiotics against ribosome assembly; antibiotic discovery; and minimal cells. Furthermore, the disclosed methods enable the ability to repurpose the translational apparatus by evolving ribosomes to synthesize sequence-controlled polymers containing D-α-amino acids, β- and γ-amino acids, and, most ambitiously, polyketides. This achievement will ultimately allow the template-guided biosynthesis and evolution of sequence-controlled peptide mimetics, polyketides, fatty acids, and ever more complex molecules that combine these disparate functional units. Additionally, the disclosed methods enable evolution of ribosomes to produce new types of sequence-defined polymers that include: new catalytic triads; unique metal site; protease resistance in peptides and proteins; libraries of mixed peptide.PK hybrids; and libraries of mixed peptide.NP conjugates. Further, one could use the disclosed methods to test and engineer and/or modify the ribosome to produce polymers based on novel poly-condensation chemistries.
The advantages of the disclosed methods are several. The disclosed methods improve upon existing ribosome engineering and modifying approaches by using a wholly in vitro ribosome evolution method. This is the first method to our knowledge that allows for whole 70S ribosome evolution without dominant lethal constraints. The disclosed ribosome evolution method shows greater than 1,000-fold specificity for functional ribosomes under different conditions. The in vitro ribosome evolution method allows for rapid probing of viability of rRNA sequence modifications. It could also allow researchers to understand the fundamental constraints for engineering and modifying the RNA based active site of the ribosome and the polymer excretion tunnel.
The method of ribosome engineering and modifying outlined herein is of great interest to the fields of biotechnology, chemistry, and material science. Previous approaches have depended on in vivo ribosome biogenesis. Yet in vivo ribosome biogenesis is limited by cell viability restrictions and transformation efficiency and requires purification of the ribosomes from cell lysates. The development of an in vitro ribosome biogenesis technology removes these limitations and expands the possibilities of ribosome engineering and modification. Ribosomes may be engineered and/or modified to incorporate unnatural amino acids for expanded protein functionality or to perform new chemistry for the production of non-protein polymers.
The disclosed methods modify iSAT technology to combine it with ribosome display to enable ribosome evolution. This may allow us to identify new methods for inhibiting the ribosome to lead to new antibiotics. In addition, evolved ribosomes may be able to synthesize sequence-controlled polymers containing D-α-amino acids, β- and γ-amino acids, and, most ambitiously, polyketides. This achievement will ultimately allow the template-guided biosynthesis and evolution of sequence-controlled peptide mimetics, polyketides, fatty acids, and ever more complex molecules that combine these disparate functional units. Further, it will allow the manufacture of polymers based on alternative poly-condensation chemistries (i.e., non amide bonds).